Methods, devices, and systems for the fabrication of materials and tissues utilizing electromagnetic radiation

ABSTRACT

The present invention provides a three-dimensional bioprinter for fabricating cellular constructs such as tissues and organs using electromagnetic radiation (EMR) at or above 405 nm. The bioprinter includes a material deposition device comprising a cartridge for receiving and holding a composition which contains biomaterial that cures after exposure to EMR. The bioprinter also includes an EMR module that emits EMR at a wavelength of about 405 nm or higher. Also provided is a bioprinter cartridge which contains cells and a material curable at a wavelength of about 405 nm or greater. The cells are present in a chamber and are extruded through an orifice to form the cellular construct.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the priority of U.S. ProvisionalPatent Application No. 61/969,832, filed Mar. 25, 2014 and U.S.Provisional Patent Application No. 62/046,279 filed Sep. 5, 2014, whichapplications are incorporated by reference.

FIELD OF THE INVENTION

The disclosed invention is in the field of bioprinting materialsutilizing electromagnetic radiation.

BACKGROUND

In today's age, machines have completely changed lives, ranging from thefirst computer to cellphones. However, the most precise andwell-articulated systems remain those that nature has built. The humanbody is an example of one such system which remains to be re-engineered.

Organ transplantation has existed since the mid-1800s when the firstskin transplant was performed. Since this time, transplantation hasexploded, resulting in the transplantation of an organ or limb or evenseveral organs/limbs simultaneously. Initially, organs only from livingidentical twins were transplanted. However, soon thereafter organs weretransplanted from the living and deceased, providing that the patientand donor have close genetic similarities. The donors could be a familymember or even a genetically compatible stranger. In fact, more than600,000 transplants have occurred in the United States since 1988.

The quest for donor tissues and organs is a slow and uphill battle.Simply stated, there are not enough donor tissues and organs and morethan 6,000 people die each year due to organ failure. There arepresently over 120,000 people in the US alone on waiting lists fororgans and others experiencing chronic problems due to the long-termdamaging effects of post-transplant immunosuppression. This has promptedsignificant research and tests on fabricating mechanical organs andtransplanting tissue and organs from non-humans, neither of which hashad much success. Unfortunately, the need for donor tissues and organshas also resulted in the black-market sale of tissues and organs fromboth willing and unwilling individuals.

Donor tissues, organs, and even animals are also used in the testing andevaluation of pharmaceutical drugs. In fact, in bringing apharmaceutical drug to the market, it takes years, even decades ofanimal testing before clinical trials on humans may be performed. Notonly do some have the view that animal testing is inhumane, but it isexpensive and inefficient, particularly in situations where thepharmaceutical drug fails to make it to market.

Animal tissues and organs are incredibly complex, possessing multipledifferent compartments that communicate with each other, intricatemicroarchitecture within these compartments, and many different celltypes within each compartment. Bioprinting involves recreating the 3Dstructure of a tissue using a fabrication technique where a computerprogram slices up a construct into discrete layers and rebuilds themusing a biomaterial. These biomaterials are designed to mimic thearchitecture of the extracellular matrix in which cells are suspended.Additionally, cells themselves can be incorporated into theseconstructs. Accordingly, a complex organ may be built step-by-step usingthe 3D images, such as those from MRI and CT scans, native cells from apatient, and biologically compatible materials.

Thus, there is a need for devices, systems, and methods for bioprintingtissues and organs, without the need for donor organs in transplantationsurgeries and animal testing in a number of industries. The invention isdirected to these and other important needs.

SUMMARY OF THE INVENTION

In one aspect, a three-dimensional bioprinter is provided and includes amaterial deposition device comprising a cartridge for receiving andholding a composition containing biomaterial that cures after exposureto electromagnetic radiation (EMR) at or above 405 nm. The bioprinteralso includes an EMR module that emits EMR at a wavelength of about 405nm or higher.

In another aspect, an EMR module for a bioprinter is provided andincludes an EMR source that emits EMR at or above 405 nm and exposes acomposition to EMR.

In a further aspect, a cellular construct prepared using the bioprinteras described herein is provided.

In yet another aspect, a tissue construct is provided and contains anEMR responsive material and cells. The tissue construct is depositedusing a bioprinter as described herein, exposing it to EMR at awavelength of about 405 nm or greater.

In still a further aspect, a bioprinter cartridge containing cells and amaterial curable at a wavelength of about 405 nm or greater is provided.

In another aspect, a bioprinter cartridge is provided and includes (i) achamber holding cells and a material curable at an EMR wavelength ofabout 405 nm or greater and an (ii) orifice through which the cells andmaterial are extruded.

In a further aspect, a method for forming an array of cells is provided.The method includes supplying a composition containing biomaterial to acartridge having an orifice through which the composition flows. Thecomposition cures after exposure to EMR of a wavelength of about 405 nmor greater. The composition flows through the orifice onto a substrate.

In yet another aspect, a method of fabricating a tissue construct isprovided. The method includes depositing a composition onto a support,wherein the composition contains cells and at least one extrusion agentwhich cures after exposure to EMR of a wavelength of about 405 nm orgreater. The method also includes curing and/or incubating thecomposition for about 1 minute to about 1 year.

In still a further aspect, a kit is provided and includes (i) a firstextrusion agent, (ii) a photo-initiator, and (iii) a second extrusionagent. The kit may also include a biomaterial.

In another aspect, a method of testing a chemical agent is provided andincludes (i) applying the chemical agent to a cellular structureprepared using the bioprinter described herein; and (ii) measuring theviability of the cells in the cellular structure.

In a further aspect, a method for transplanting a synthetic organ in amammal is provided and includes transplanting a cellular constructprepared using the bioprinter described herein to the mammal.

The general description and the following detailed description areexemplary and explanatory only and are not restrictive of the invention,as defined in the appended claims. Other aspects of the presentinvention will be apparent to those skilled in the art in view of thedetailed description of the invention as provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The summary, as well as the following detailed description, is furtherunderstood when read in conjunction with the appended drawings. For thepurpose of illustrating the invention, there are shown in the drawingsexemplary embodiments of the invention; however, the invention is notlimited to the specific methods, compositions, and devices disclosed. Inaddition, the drawings are not necessarily drawn to scale. In thedrawings:

FIG. 1 depicts an embodiment of the bioprinter described herein havingan emitter module and receiving plate.

FIG. 2 illustrates an embodiment of 3D bioprinter described herein thathas a 3 axis system on which devices can move.

FIG. 3 illustrates a connecting means for connecting a cartridge to abioprinter described herein.

FIG. 4 is one embodiment of a cartridge containing one or more EMRmodule described herein.

FIG. 5 is an embodiment of a cartridge described herein that holds oneor more compositions described herein.

FIG. 6 illustrates a connecting means holding a cartridge for abioprinter described herein.

FIG. 7 illustrates an embodiment of a cartridge used in the methods,systems, and devices described herein.

FIG. 8 illustrates an embodiment of fused deposition manufacturing via amultiple layering technique.

FIG. 9 illustrates an embodiment of the invention using multiple syringeheads.

FIG. 10 illustrates an embodiment of an apparatus described herein whichwas used to create 3D tissue structures.

FIG. 11 illustrates one material deposition device having an EMR emittermodule directing electromagnetic radiation (EMR) over a broad areatowards the receiving plate.

FIG. 12 depicts one material deposition device having an EMR emittermodule directing the EMR to a specific location on the receiving plateto interact with the material upon, during, or after deposition.

FIG. 13 illustrates an embodiment of a material deposition device havingan EMR emitter module directing the EMR within the material devicecompartment to begin solidify the material before deposition andinteraction with the receiving plate.

FIG. 14A illustrates a three-dimensional cell patterning of a twodifferent materials to produce a single composite structure. FIG. 14Bdepicts a cross sectional view of the structure and FIG. 14C depicts atop view of the structure demonstrating the lattice structure of thelayered materials.

FIG. 15 is a diagram illustrating embodiments of compounds utilized toprepare the compositions described herein.

FIG. 16 is a diagram illustrating an embodiment of using multiplecartridges and the contents of two syringes.

FIG. 17 is a bowl construct designed on the 3D CAD Solidworks® software.

FIG. 18 is a photograph of a cell-based bowl prepared using the methodsand systems described herein.

FIG. 19 is an ear construct designed on the 3D CAD Solidworks® software.

FIG. 20 is a photograph of a synthetic ear prepared using the methodsand systems described herein.

DETAILED DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS

The present invention may be understood more readily by reference to thefollowing detailed description taken in connection with the accompanyingfigures and examples, which form a part of this disclosure. It is to beunderstood that this invention is not limited to the specific devices,methods, applications, conditions or parameters described and/or shownherein, and that the terminology used herein is for the purpose ofdescribing particular embodiments by way of example only and is notintended to be limiting of the claimed invention. Also, as used in thespecification including the appended claims, the singular forms “a,”“an,” and “the” include the plural, and reference to a particularnumerical value includes at least that particular value, unless thecontext clearly dictates otherwise. The term “plurality,” as usedherein, means more than one. When a range of values is expressed,another embodiment includes from the one particular value and/or to theother particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another embodiment. All ranges areinclusive and combinable.

It is to be appreciated that certain features of the invention whichare, for clarity, described herein in the context of separateembodiments, may also be provided in combination in a single embodiment.Conversely, various features of the invention that are, for brevity,described in the context of a single embodiment, may also be providedseparately or in any subcombination. Further, reference to values statedin ranges includes each and every value within that range.

The present invention may be understood more readily by reference to thefollowing description taken in connection with the accompanying Figuresand Examples, all of which form a part of this disclosure. It is to beunderstood that this invention is not limited to the specific products,methods, conditions or parameters described and/or shown herein, andthat the terminology used herein is for the purpose of describingparticular embodiments by way of example only and is not intended to belimiting of any claimed invention. Similarly, unless otherwise stated,any description as to a possible mechanism or mode of action or reasonfor improvement is meant to be illustrative only, and the inventionherein is not to be constrained by the correctness or incorrectness ofany such suggested mechanism or mode of action or reason forimprovement. Throughout this text, it is recognized that thedescriptions refer both to the features and methods of making and usingthe coatings and films described herein.

In the present disclosure the singular forms “a,” “an,” and “the”include the plural reference, and reference to a particular numericalvalue includes at least that particular value, unless the contextclearly indicates otherwise. Thus, for example, a reference to “amaterial” is a reference to at least one of such materials andequivalents thereof known to those skilled in the art, and so forth.

When a value is expressed as an approximation by use of the descriptor“about” or “substantially” it will be understood that the particularvalue forms another embodiment. In general, use of the term “about” or“substantially” indicates approximations that can vary depending on thedesired properties sought to be obtained by the disclosed subject matterand is to be interpreted in the specific context in which it is used,based on its function. The person skilled in the art will be able tointerpret this as a matter of routine. In some cases, the number ofsignificant figures used for a particular value may be one non-limitingmethod of determining the extent of the word “about” or “substantially”.In other cases, the gradations used in a series of values may be used todetermine the intended range available to the term “about” or“substantially” for each value. Where present, all ranges are inclusiveand combinable. That is, references to values stated in ranges includeevery value within that range.

When a list is presented, unless stated otherwise, it is to beunderstood that each individual element of that list and everycombination of that list is to be interpreted as a separate embodiment.For example, a list of embodiments presented as “A, B, or C” is to beinterpreted as including the embodiments, “A,” “B,” “C,” “A or B,” “A orC,” “B or C,” or “A, B, or C.”

It is to be appreciated that certain features of the invention whichare, for clarity, described herein in the context of separateembodiments, may also be provided in combination in a single embodiment.That is, unless obviously incompatible or excluded, each individualembodiment is deemed to be combinable with any other embodiment(s) andsuch any combinations is considered to be another embodiment.Conversely, various features of the invention that are, for brevity,described in the context of a single embodiment, may also be providedseparately or in any subcombination. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.Finally, while an embodiment may be described as part of a series ofsteps or part of a more general structure, each said step may also beconsidered an independent embodiment in itself.

“Bioprinting” as used herein a three-dimensional, precise deposition ofcells using an automated, computer-aided, three-dimensional prototypingdevice (e.g., a bioprinter).

The bioprinted cellular constructs, tissues, and organs are preparedusing methods utilizing a rapid prototyping technology based onthree-dimensional, automated, computer-aided deposition of cells.Advantageously, the bioprinters described herein are capable ofgenerating organs and the like which do not result in an immune responseand thus will not require the administration of an immunosuppressant fortransplantation of an organ. The bioprinters also are uncontaminated andwill not comprise any infectious agents such as viruses, bacteria, orthe like. The bioprinters completely obviate the need for donor organsand are even cost effective since the organs are prepared frominexpensive biomaterials. The bioprinters also reduce or eliminate theneed for animal testing of any new chemical, including pharmaceuticalagents.

A. BIOPRINTER

The bioprinter may include any instrument that automates the bioprintingprocess described herein. In one embodiment, the bioprinter is a 3Dprinter, which may be selected by one skilled in the art. Any componentof the bioprinter described herein may be operated by manual or roboticmeans as determined by one skilled in the art.

The bioprinter contains one or more rods housed within the interior. Inone embodiment, the rods may be placed at any direction or height and beof any width that is necessary to support one or more component of thebioprinter. In a further embodiment, the rods are placed along thex-axis, y-axis, or z-axis, or any combination thereof in the bioprinter.In another embodiment, the cartridge, receiving means, printer stage, orany combination thereof is attached to one or more rod. In a furtherembodiment, the cartridge moves along the x and y rods and the printerstage moves along the z rod.

The rods permit the receiving means to remain at the height needed tofabricate the article. The rods may also be utilized to calibrate and/orlevel one or more component of the bioprinter. In one embodiment, therods control movement of one or more component of the bioprinterincluding, without limitation, the cartridge, printer stage, or anycombination thereof. The movement of the rods may be performed usingskill in the art including, without limitation, a motor.

The rods housed within the bioprinter may also include endstops. Theendstops are a means of defining a boundary to build the fabricatedarticle. The endstops are also useful to keep one or more component ofthe bioprinter in a particular position. The endstops may contribute tocalibrating the position of one or more component on the respective x,y, and/or z rod. In one embodiment, the endstops ensure that thecartridge stays within the area of the receiving means. In anotherembodiment, the x and y endstops define the boundary for the cartridge.Accordingly, the x and y endstops restrict the movement of the cartridgeto the size of the receiving means. For example, the cartridge may hitan endstop and cannot proceed past this point, i.e., it stays within thearea of the receiving means. In a further embodiment, the z endstopdefines the boundary for the receiving means and/or printer stage.Accordingly, the z endstop assists in modulating the height of thereceiving means. In this instance, the z endstop ensures that theprinter stage and receiving means do not move too high. In doing so, thez endstop may prevent the receiving means from contacting the needle anddamaging the syringe and/or destroying the fabricated article. Theendstops may be fabricated using any materials available in the artincluding, without limitation, glass, coated glass, plastic, coatedplastic, metal, a metal alloy, gel, or any combination thereof.

As noted above, one or more component of the bioprinter may becalibrated prior to or at one or more times during the bioprinting.Accordingly, the bioprinter contains a calibrating means for obtainingthe proper level for one or more component. In one embodiment, one ormore of the cartridge, printer stage, receiving means, among others, iscalibrated. In another embodiment, one or more component of thebioprinter is calibrated along one or more of the x, y, and z axes.Calibration of the bioprinter may be performed using manual techniques,automated techniques, or a combination thereof. In one embodiment, thecalibration means may include laser alignment, optical alignment,mechanical alignment, piezoelectric alignment, magnetic alignment,electrical field or capacitance alignment, ultrasound alignment, or anycombination thereof.

The procedure for calibrating one or more component of the bioprinterincludes use of the above-noted rods. The one or more component of thebioprinter is attached to one or more rod by one or more screw. In oneembodiment, the receiving means and the printer driver are attached tothe same rod or rods. In another embodiment, the receiving means andprinter driver are attached to a single x axis rod. In a furtherembodiment, the receiving means and printer driver are attached to therod using one or more screw. In yet a further embodiment, the receivingmeans and printer driver are attached to the rod using three screws. Instill another embodiment, one or more screws pass through the receivingmeans and printer driver and are secured on the underface of the printerdriver. In yet a further embodiment, the one or more screw is securedusing means in the art including a wingnut. To facilitate theadjustment, a spring may be placed between the receiving means andprinter drive. The selection of the spring is within skill in the art.In one embodiment, the spring is metal, plastic, or the like. In anotherembodiment, the spring is zinc plated. In a further embodiment, thespring is a zinc plated music wire. The length and diameter of thespring depends on the size of the bioprinter, components therein, andarticle being fabricated. In one embodiment, the length of the spring isabout 1 mm to about 75 mm. In a further embodiment, the length of thespring is about 10 to about 30 mm. In another embodiment, the diameterof the spring is about 1 to about 10 mm. In yet a further embodiment,the screw passes through the spring. The location of the receiving meansalong the x, y, and z axes may be adjusted by tightening and loosening,i.e., screwing or unscrewing, the wingnuts.

In order to prepare the fabricated materials, the bioprinters disclosedherein dispense the composition with repeatable accuracy. In oneembodiment, the position of the cartridge is calibrated along thex-axis, the y-axis, and the z-axis, or any combination thereof. Theaccuracy is dependent on a number of factors, including, withoutlimitation, removal and insertion of cartridges, position of thecartridge, among others. Calibrating the position of the cartridgeincludes the use of a laser, may be manual (e.g., visual), or anycombination thereof.

The atmosphere of the bioprinter may also be adjusted to provide theoptimal conditions for depositing the composition. Specifically, thetemperature, humidity, atmospheric composition, among others may bevaried. In one embodiment, the bioprinter may include a means foradjusting temperature of the bioprinter, in general, or of theindividual components therein including inside the cartridge, thereceiving means, or the atmosphere of the bioprinter in general. Theselected temperature may be selected by one skilled in the art and maydepend on the type of cell being printed. In one embodiment, thetemperature is maintained at a temperature which results in a suitablephysical environment for the cells. In one embodiment, the temperatureis maintained at about −10 to about 300° C. In a further embodiment, thetemperature is maintained at about 0 to about 100° C. In anotherembodiment, the temperature is maintained at about room temperature. Inanother embodiment, the temperature is maintained at about 37° C. Themeans may include a heating or cooling element. Heating elementsinclude, without limitation, radiant, convection, conductive, fan, heatexchange heater, or any combination thereof. The cooling element mayincluding, without limitation, coolant, chilled liquid, ice, a radiantcooler, convection cooler, a conductive cooler, a fan cooler, or anycombination thereof.

The humidity of the bioprinter in general or of the individualcomponents including inside the cartridge may also be varied asdiscussed above. Specifically, the humidity may be increased ordecreased as necessary. Humidities ranging from about 0% to about 100%may be utilized.

The gaseous composition of the bioprinter, when sealed, further may bevaried. In doing so, gases aside from air including varyingconcentrations of carbon dioxide, nitrogen, argon, and oxygen may beutilized and varied as needed.

Also contemplated is a means for applying a wetting agent to one any oneor more part of the bioprinter as described below including, withoutlimitation, the receiving means, cartridge, cartridge contents, or anycombination thereof. The “wetting agent” includes a fluid whichfacilitates extrusion of the composition described herein, prevents thefabricated article from adhering to the receiving means, among others.In one embodiment, the wetting agent is hydrophilic or hydrophobic. Inanother embodiment, the wetting agent is, without limitation, water,tissue culture media, buffered salt solutions, scrum, or any combinationthereof. The wetting agent may be applied before, simultaneously, orafter the composition is dispensed.

B. PRINTER STAGE

A printer stage is another component of the bioprinter described herein.The printer stage as used herein regulates the movement of the receivingmeans, as described above. In one embodiment, the printer plate movesthe receiving means up and down. The printer stage may be, withoutlimitation, glass, coated glass, plastic, coated plastic, metal, a metalalloy, gel, or any combination thereof. In one embodiment, the printerstage is square, circular, triangular, oval, rectangular, or irregularlyshaped.

The printer stage is located within the bioprinter and adjacent to thereceiving means. In one embodiment, the printer stage is positionedbelow the receiving means.

C. RECEIVING MEANS

The bioprinter is capable of dispensing composition in predeterminedgeometries, i.e., positions, patterns, layers etc., in two or threedimensions, onto a means for receiving the composition. In oneembodiment, the receiving means is a receiving plate. In anotherembodiment, the receiving means is a 3-D structure such as a limb,organ, tissue, gel, multi-well plate, or any combination thereof. In afurther embodiment, the receiving means is a water bath. In oneembodiment, FIG. 1 is a top view of a bioprinter 100 described herein.Cartridge 102 includes a means for receiving and holding a composition.EMR module 106 emits EMR 108, exposing the contents on receiving plate110. Receiving plate 110 receives the deposited material.

Another embodiment of a bioprinter described herein is presented in FIG.2. FIG. 2A is an edge-view of the bioprinter illustrating a 3 axissystem on which devices can move. FIG. 2B is a side-view of thebioprinter having cartridge 102 and receiving plate 110. This printer isprogrammed to move in 3D space by zeroing itself with endstops 118 and120. Receiving plate 110 can be leveled using a spring-based system 126and printer driver 124. For example, in this case, the bioprinter usespneumatic pressure 210 directed through conduit 128 to extrudematerials. This pressure is controlled manually by dial 130. FIG. 2C isa top-view of the bioprinter of FIG. 2B. FIG. 2D is a second side-viewof the bioprinter, but showing endstop 122. FIG. 2E is 180° side-view ofthe bioprinter of FIG. 2D.

Accordingly, the bioprinter achieves a particular geometry of thefabricated article by moving the cartridge relative to a receivingmeans. Alternatively, the receiving means is moved relative to thecartridge.

In an effort to reduce contamination, the receiving means is non-toxicto the biomaterial, components of the composition, or any combinationthereof. The locations at which the bioprinter deposits the compositiononto a receiving means are adjustable as determined by the user.

The receiving means is desirably designed specifically to accommodatethe shape, size, texture, or geometry of the fabricated article. It maybe may be flat or substantially flat; smooth or substantially smooth;defined or substantially defined; or any combination thereof. Thereceiving means may assume a variety of concavities, convexities, ortopographies based on the article to be fabricated. The receiving meansmay contain, without limitation, glass, coated glass, plastic, coatedplastic, metal, metal alloy, gel, or any combination thereof. Thereceiving means and the biomaterial may be biocompatible. In oneembodiment, the receiving means is a substantially flat plate,multi-well plate such as a 6- or 96-well plate, or 3D scaffold in whichthe cartridge moves 3 dimensionally. In another embodiment, thereceiving means is square, circular, triangular, oval, rectangular, orirregularly shaped.

The receiving means is located within the bioprinter and adjacent to thecartridge. The receiving means may also be adjacent to the printerstage. In one embodiment, the receiving means is positioned below thecartridge. In another embodiment, the receiving means is positionedabove the printer driver. In a further embodiment, the receiving meansis positioned between the cartridge and the printer stage.

The receiving means may be leveled prior to deposition of thecomposition. The leveling may be performed as described above byadjusting the printer stage using the rods and endpoints. Alternatively,the bioprinter could have a self-leveling means, thereby eliminating theneed for human intervention for leveling the hardware. In doing so,software may be used to analyze the position of the receiving means andperform any necessary adjustments. In one embodiment, the receivingmeans is leveled to 0° relative to the flat bottom of the cartridge.

D. CARTRIDGE

A “cartridge” is an object that is capable of receiving and holding acomposition prior to deposition described herein. The cartridge may beattached to the bioprinter using any means known in the art. Any numberof cartridges may be utilized and depends on the desired article forfabrication. In one embodiment, the cartridge is attached to thebioprinter through one of the aforementioned rods. In anotherembodiment, the cartridge is attached to a center piece which isattached to one or more rod. In a further embodiment, the cartridge isattached to a center piece along the x-rod.

In one embodiment, one cartridge is utilized. In this instance, all ofthe components of the composition are combined in one cartridge.

In another embodiment, two or more, i.e., multiple cartridges may beutilized. In a further embodiment, 2 to about 25 cartridges may be used.In this instance, each cartridge contains the same composition ordifferent compositions. For example, if using two cartridges, onecomposition may be deposited separately from the other composition byusing a second cartridge. By doing so, the simultaneous or separate useof multiple cartridges may be used to create complex, hierarchicalstructures.

The cartridge may be attached to one or more additional cartridge.Alternatively, the cartridge is position separately from the othercartridges.

The cartridge is made from any material which may be used in thebioprinter described herein. In one embodiment, the cartridge is glass,plastic, metal, gel, or any combination thereof. The cartridge may becoated on its interior or exterior with a casing. The casing may be madefrom any material that is compatible with the cartridge and includesglass, metal, plastic, or any combination thereof. The casing may be thesame material as the cartridge or different.

The cartridge is of any shape which fits into the bioprinter and may beselected by one skilled in the art. In one embodiment, the cartridge iscylindrically shaped. In another embodiment, the cartridge is graduatedat one end, i.e., one end is triangularly shaped and conicals downward.FIG. 3 illustrates a center piece 180 for attaching cartridge 102 to abioprinter described herein. Cartridge 102 is cylindrically shaped andcontains grooves for insertion of cartridge 102 and orifice 182.Cartridge 102 is slid into center piece 180 along axis 184.

The cartridge contains a chamber and at least two openings. Thecartridge has a capacity which is dependent of the selected fabricatedarticle, composition, size of the dispensing means, among others. In oneembodiment, the cartridge has a diameter of about 1 μm to about 10 mm.In another embodiment, the cartridge has a diameter of about 1 to about10 mm. In a further embodiment, the cartridge has a capacity of at leastabout 0.1 mL. In another embodiment, the cartridge has a capacity ofabout 0.1 mL to about 5000 mL. In still a further embodiment, thecartridge has a capacity of about 1 mL to about 100 mL. In yet anotherembodiment, the cartridge has a capacity of about 1 to about 20 mL.

In one embodiment, the cartridge contains one opening at one end and asecond opening at the opposite end. In another embodiment, the cartridgecontains one opening which permits insertion of a dispensing means intothe chamber. In a further embodiment, the cartridge contains a secondopening which permits a portion of a dispensing means, i.e., the needle,to exit the cartridge. The size of the first and second openings dependson the dispensing means utilized in fabrication of the article. In oneembodiment, the first and second openings are, independently, about 1 μmto about 10 cm. In another embodiment, the first and second openingsare, independently, about 2 to about 10 mm. FIG. 4 depicts a cartridgethat holds and deposits the composition. FIG. 4A is cartridge 102containing a composition containing biomaterials 166. The upper portion174 of cartridge 102 generates the force, current, or temperaturedifferential to permit deposition of the composition through extrusionorifice 176. FIG. 4B is a second cartridge 102 containing the componentsof FIG. 4A, but having a solid material 178 contained therein.

The cartridge also may be open to atmospheric conditions of the room orclosed to atmosphere conditions (i.e., open only to the atmosphericconditions of the bioprinter). Any opening of the cartridge may betemporarily or permanently sealed. The cartridge is modifiable to holddifferent dispensing means. The tip of the dispensing means may beoptionally capped to seal the components of the dispensing means fromatmospheric pressure. In one embodiment, the cartridge and/or dispensingmeans is a closed system, i.e., limiting the exposure of the user to thespecific contents in the cartridge. In another embodiment, the cartridgeand/or dispensing means is an open system for compositions that aresufficiently viscous to drive deposition without the need for exogenousmethods. In one embodiment, the cartridge is sealed using a cap or lidwhich adaptable to the particular cartridge being utilized. In anotherembodiment, the lid provides the mechanism for attaching the cartridgeto the bioprinter. Accordingly, the cap or lid may include a firstportion which attaches to and seals the cartridge and a second portionwhich attaches to a bioprinter. In one embodiment, the lid attaches to acenter piece of the bioprinter.

The center piece of the bioprinter, as noted above, secures thecartridge to the bioprinter. The cartridge is designed so as to bephysically compatible with the center piece and contains an opening intowhich the cartridge may be placed/inserted.

The cap of the cartridge may attach directly to the cartridge of mayattach thereto via one or more cap holders. The cap holder(s) attach tothe cartridge. The cap is also compatible with the cap holder andsecurely fit together to substantially seal the cartridge. In oneembodiment, the cap, cap holder, and the cap/cap holder secured togetherhave grooves and ridges, i.e., a specific shape. Conversely, the centerpiece has the opposite grooves and ridges to that of the cap, capholder, and/or cap/cap holder secured together. FIG. 5 is one embodimentof a cartridge of the bioprinter described herein. Cartridge 102contains casing 200, two cap holders 202, and the lid/cap 132 that locksthe syringe in place. The bottom of the cartridge contains two LEDs 204that are housed inside compartments. The LEDs are connected in seriesand are controlled by an LED driver 206.

FIG. 6 depicts center piece 180 directly adjacent to cartridge 102 ofFIG. 5. Compression 186 creates a seal between center piece 180 andcartridge 102 to ensure proper deposition of the inner contents. Amanual or automated system 188 creates the connection between cartridge102 and center piece 180.

The cartridge is secured into the center piece using skill known in theart. In one embodiment, the cartridge is secured into the center pieceusing mechanical force, electromagnetic force, or pressurized force. Inanother embodiment, the cartridge is secured into the center piece usingone or more latch. In a further embodiment the cartridge is secured intothe center piece using magnetic attraction, collet chuck grip, ferrule,nut, barrel adapter, or any combination thereof. The cartridge may beclipped or snapped in (manually or with magnetic force) or a robotic armcan be used to replace each cartridge in the limited number ofcartridges as the printing proceeds. Compression may be applied to thecenter piece, cartridge, or any combination thereof to create a seal. Inone embodiment, the seal prevents unwanted gases or solid particles fromentering the cartridge. In another embodiment, the seal assists in thedeposition of the composition. The compression may be applied manuallyor may be automated.

The bioprinter may also include a sensing means for sensing if thecartridge is locked into the center piece. In one embodiment, thesensing means is a magnetic sensor, electrical signal, mechanicalswitch, or any combination thereof. The sensing means may furtherinclude an alert if the cartridge is not locked into the center piece.In one embodiment, the sensing means is a light sensor, alarm, or anycombination thereof. In another embodiment, the alert is generated usinga light gate or motion sensor.

The cartridge may be permanently or temporarily marked (pen or sticker),colored, dyed, scored, painted, polished, or any combination thereof.The cartridge may be uncovered, partially covered or fully covered usingany means known in the art. In one embodiment, the cartridge prevent thecontents therein from being prematurely exposed to the EMR (i.e.,exposed to light). In another embodiment, the cartridge is covered topresent premature EMR exposure. In a further embodiment, the cartridgeis impermeable to light having a wavelength of about 405 nm or greater.In doing so, the covering prevents the composition from curing in thecartridge and jamming the dispensing means, i.e., the syringe. Any partof the cartridge may be covered including, without limitation, theentire cartridge, the tip of the cartridge, a portion of the cartridge,or any combination thereof. In another embodiment, the cartridge iscovered using aluminum foil, adhesive foil, a plastic film such as aParafilm® coating, or the like.

E. DISPENSING MEANS

The cartridge utilized herein houses and protects a dispensing means.Many types of dispensing means are suitable for use with bioprintersdisclosed herein and the methods of using the same. One of skill in theart would recognize that different dispensing means are required fordifferent compositions containing biomaterial. For example, certaincompositions may degrade plastic and, in that case, glass or metaldispensing means may be used.

The dispensing means contains one or more orifice through which thecomposition exits. In one embodiment, the dispensing means contains asingle orifice. The orifice must be large enough to permit dispensing ofthe composition, but not too large as to have uncontrolled dispensing ofthe composition. The shape of the orifice is not a limitation and maybe, without limitation, flat, circular, square, rectangular, triangular,oval, polygonal, irregular, smooth or textured. Accordingly, selectionof a suitable orifice depends on the components and viscosity of thecomposition. In one embodiment, the orifice has a diameter of about 1 toabout 1000 or more μm. In another embodiment, the orifice has a diameterof about 1 μm to about 100 μm.

The dispensing means may be a capillary tube, a micropipette, syringe ora needle. In one embodiment, the dispensing means contains a needlehaving a luminal diameter of about 10μ to about 5 cm. In anotherembodiment, the dispensing means contains a needle having a luminaldiameter of about 1 mm to about 10 mm. In a further embodiment, thedispensing means contains a needle of about 1 mm to about 300 mm inlength. In yet another embodiment, the needle is about 10 to about 100mm in length. In still a further embodiment, the dispensing means is aLuer-Lok® Tip sterile syringe. In another embodiment, the dispensingmeans has a ⅕ mL graduation. In a further embodiment, the dispensingmeans has an about 6 mm (0.25″) high precision tip

FIG. 7 displays cartridge 102 used in this specific device. A syringe112 is used to hold the composition for extrusion. Cap 132 is used toseal off syringe 112 from the atmospheric pressure and pneumaticpressure is transmitted to syringe 112 contents 134 using compressed airthrough conduit 128. The composition 134 within syringe 112 is depositedonto a receiving plate using EMR 108 from EMR module 106 into tissuestructure 136.

The embodiment presented in FIG. 8 illustrates the deposition ofcomposition 134 layer by layer to create a 3D tissue via cartridge 102.EMR 108 chemically transforms composition 134 from a liquid state into agel or solid state and binds layers together to create the construct.Air driven through conduit 128 puts pressure on composition 134 withinsyringe 112. Material is deposited layer-by-layer (layer 137, layer 138,layer 139) until tissue structure 140 is completed.

FIG. 9 illustrates an embodiment of using multiple cartridges andsyringes for extrusion. Multiple cartridges 102 and 104 are used tocreate tissue 142. Support material 148 from cartridge 102 is printed inlayers and cells encapsulated in a second composition 146 in the nextlattice or layer. Support material 148 hardens with EMR 108. Cartridge102 with cell-laden composition 148 deposits its contents on receivingplate 110 and is solidified using EMR 108.

The contents of the dispensing means may be optionally primed prior touse to increase the accuracy of the process. The priming includes makingthe contents of the dispensing means ready to be dispensed.

The dispensing means may be disposable or permanent. In one embodiment,the dispensing means is ejected or removed, automated or manually, fromthe bioprinter following extrusion, dispensing, or deposition of thecontents. In another embodiment, a new dispensing means is attached tothe bioprinter. In a further embodiment, the cartridge is a premixed andpre-sealed cartridge which contains the necessary composition. By doingso, the user may purchase the cartridge and would not need to refill thedispensing means by preparing and adding the composition.

The dispensing rate of the dispensing means is dependent on one or morefactors as determined by those skilled in the art. In one embodiment,the dispensing rate is dependent on the viscosity of the composition. Inanother embodiment, the dispensing rate is dependent on the pressureapplied to the composition. In a further embodiment, the dispensing rateis high so that a fine line of composition may be deposited. In yetanother embodiment, the dispensing rate is low so that a thicker line ofcomposition may be deposited.

The dispensing means may be sealed for ease of use or to avoidcontamination of the contents therein. Alternatively, the dispensingmeans are not sealed and may be opened by the user. In one embodiment,the dispensing means is sealed using cap which is permanently affixed tothe dispensing means and cannot be pierced using a needle or the like.In another embodiment, the dispensing means is sealed using a cap whichis permanently affixed to the dispensing means, but the cap may bepierced with a needle by the user. In a further embodiment, thedispensing means is sealed using a cap which may be easily removed bythe user. In another embodiment, the dispensing means is impermeable toEMR of a wavelength of about 405 nm or greater.

F. EXTRUSION MEANS

The composition passes through the dispensing means using systems knownin the art. In one embodiment, the composition is deposited onto thereceiving means using gravity. In another embodiment, deposition of thecomposition may be facilitated via the use of an extrusion means. Theterm “extrude” or variations thereof as used herein refers to theability of the composition to be forced to exit the dispensing means.

As one option, the extrusion means is a pressure means for controllingthe pressure provided to the cartridge, dispensing means, or anycombination thereof. The pressure may be generated using any systemknown in the art including, without limitation, pneumatic systems usingcompressed gas such as compressed air, argon, carbon dioxide, ornitrogen, hydraulics, pistons, screw-based means, or any combinationthereof. The pressure required to deposit the composition depends on thearticle to be fabricated and contents of the composition, among others.In one embodiment, the pressure is about 50 to about 1500 kPa (about 0.1to about 150 psi). In one embodiment, the compressor which directs thegas at the dispensing means and/or cartridge is connected to andoperatively associated with the cartridge. By doing so, a controller andpressure pump is provided for the dispensing means. The pressure fromthe compressor drives deposition of the composition onto the receivingmeans. The pressure may be controlled using a dial operatively connectedto the compressor. If more than one compressor is used, one dial maycontrol the pressure of the compressor(s) or two or more dials may beutilized in an effort to obtain different pressures in differentcartridges. In one embodiment, the compressed gas is fed into thecartridge and/or syringe using a hose. Each cartridge may utilize thesame pressure to dispense the contents therein or use varying pressures.

FIG. 10 is a schematic of a system and apparatus provided herein. Thesystem includes computer 114, air compressor 116, and bioprinter 100.Air compressor 116 is connected to and operatively associated withcartridge 102 to provide a controller and pressure pump for the syringe.The pressure from air compressor 116 drives deposition of biomaterialonto receiving plate 110. All can be controlled by CAD softwareprogrammed in computer 116.

The extrusion means may also be thermal, electrical, piezoelectric, ormechanical as determined by those skilled in the art. In one embodiment,heat is applied to the composition, thereby reducing its viscosity. Inanother embodiment, the composition is electrically charged using acurrent. In a further embodiment, the composition is extruded usingpiezoelectric methods. In yet another embodiment, the composition isextruded using mechanical means such as a screw system to drivedeposition.

G. EMR SOURCE

The bioprinter includes an EMR module to cure the materials. The term“electromagnetic radiation” (EMR) as used therein refers to light havinga wavelength of at least those in the visible spectrum. In oneembodiment, the EMR is light in the visible spectrum. In anotherembodiment, the EMR is light in the near-infrared (NIR) spectrum. In afurther embodiment, the EMR is in the infrared spectrum. In yet anotherembodiment, the EMR is about 405 nm or greater. In another embodiment,the EMR is about 405 nm to about 1 mm. In a further embodiment, the EMRis about 405 nm to about 700 nm. In still another embodiment, the EMR isabout 1 mm to about 750 nm. In yet a further embodiment, the EMR isabout 405 to about 410 nm.

The EMR module includes an EMR source that emits EMR at or above 405 nmand exposes a composition described herein to EMR. Many EMR sources aresuitable for use with the EMR module described herein. In oneembodiment, the EMR is a light emitting diode. In another embodiment,the EMR is an IR laser. One of more EMR source may be utilized todepending on the number of EMR modules utilized in the bioprinter. TheEMR sources may be connected in series or in parallel.

The EMR module may include a chamber adjacent to the contents of thecartridge to EMR source. The EMR module may be separate from orpermanently, semi-permanently, or reversibly attached to the bioprinter.In one embodiment, the EMR module is placed adjacent to the bioprinterso that the EMR is capable of reaching the composition. In anotherembodiment, the EMR module is physically attached or incorporated intothe cartridge, i.e., the cartridge houses the EMR module. When thecartridge houses the EMR module, it may be on one side, multiple sides,inside, or outside of the cartridge.

The EMR is tunable with respect to wavelength, intensity, exposure time,or any combination thereof. In one embodiment, each EMR may be dimmed,brightened or focused depending on the curing required by the fabricatedarticle. In a further embodiment, the EMR module contains an attenuationfilter which lowers or raises the intensity of the EMR. In anotherembodiment, the EMR module is tuned based on the curing times, size, orany combination thereof which are required by the fabricated article.The EMR module may also be oriented to focus the EMR in any number ofdirections. By doing so, the EMR is accurately focused at the requiredposition. The EMR module may be controlled using an EMR driver. In oneembodiment, the total radiance of the EMR module is about 1 to about 10mW/cm². In another embodiment, the total radiance of the EMR module isabout 5 mW/cm².

The composition is exposed to the EMR for a period of time sufficient tocure the material. Suitable exposure times include 1 or more seconds. Inone embodiment, the composition is exposed to the EMR for about 1 secondto about 1 year.

The EMR module is capable of exposing the biomaterials to EMR prior to,concurrently with, subsequent to, or any combination thereof to thedispensing. In one embodiment, the EMR is broadly directed in thevicinity of the deposition site. FIG. 11 is a side view of cartridge 102having syringe 112 and EMR module 106 directing EMR 108 broadly towardsreceiving plate 110 to interact with the material upon, during, or afterdeposition. EMR module 106 emits EMR 108 broadly exposing the contentson receiving plate 110. In another embodiment, the EMR is specificallyfocused in one area on the deposition site. FIG. 12 is another side viewof cartridge 102 having syringe 112 and EMR module 106 directing EMR 108precisely towards a specific location on receiving plate 110 to interactwith the material upon, during, or after deposition. In a furtherembodiment, the EMR is focused on at least one component of thecomposition being deposited. In yet another embodiment, the EMR isfocused on the composition as it exits the deposition device. In still afurther embodiment, the EMR module is contained within the cartridge andfocuses EMR on the composition prior to deposition. FIG. 13 is a furtherside view of cartridge 102 having syringe 112 and EMR module 106directing EMR 108 within the material device compartment to beginsolidifying the material before deposition and interaction withreceiving plate 110.

The EMR may also be sources of energy. In one embodiment, the EMR may begenerated by interacting NIR light with gold nanorods to generate heatthrough the photothermal effect. In one embodiment, this EMR isgenerated in the presence of a thermal initiator. See, Gramlich,“Transdermal Gelation of Methacrylated Macromers with Near-infraredLight and Gold Nanorods,” Nanotechnology, 25:014004, 2014 which isincorporated by reference.

There may be a single EMR module or may be several modules depending onthe other components of the bioprinter and the fabricated article to beprepared. The EMR modules may all run at the same wavelength or maydiffer.

It is also contemplated that EMR modules may pulse going from bright todim or dim to bright. In one embodiment, the EMR module pulses as eachlayer is printed.

H. OPTICAL DEVICE

The bioprinter described herein may optionally include an optical devicefor viewing the fabricated article. In one embodiment, the opticaldevice contains a lens having a blue filter. By doing so, the fabricatedarticle may be viewed and/or recorded without interference from the EMR,thereby providing increased quality control in monitoring and/orpreparing the article. In another embodiment, the optical device is anoptical recorder such as a camera, video camera, heat sensor camera, orany combination thereof. The optical device is at a resolution that isrequired for the particular composition being utilized and article beingfabricated. Accordingly, the resolution of the optical device may below, medium, or high, as determined by those skilled in the art.

The optical detector may be placed at any location of the bioprinter. Inone embodiment, the optical device is placed in close proximity to thefabricated article. In another embodiment, the optical device is mountedon one or more component of the bioprinter or is adapted to move alongside of the receiving means and/or cartridge. In a further embodiment,the optical device is mounted on the cartridge, receiving means, in thecorner of the bioprinter, among others. In another embodiment, theoptical device is mounted on the cartridge. In a further embodiment, theoptical device is mounted adjacent to the receiving means. In yet afurther embodiment, the optical device is mounted on the cartridgefacing the receiving means. In still another embodiment, the opticaldevice is adapted to move inside of the bioprinter by way of a track orthe like.

The optical device may be temporarily or permanently attached to one ormore component of the bioprinter. In one embodiment, the optical deviceis attached to the EMR module. In another embodiment, the optical deviceis permanently attached to the EMR module. In a further embodiment, theoptical device is reversibly attached to the EMR module.

I. SOFTWARE

The bioprinter deposits the composition at precise locations (in two orthree dimensions) on the receiving means. The locations are dependent onthe form being prepared and inputted information, which is translatedinto computer code. As known in the art, the computer code is a sequenceof instructions, executable in the central processing unit (CPU) of adigital processing device, and written to perform a specified task.Additional bioprinting parameters including, without limitation, heightof the cartridge, pump speed, robot speed, control of variabledispensing means, EMR exposure time, cartridge position, direction ofthe cartridge, and speed of the cartridge, among others.

Computer aided design software may be utilized to prepare the tissueconstructs. In one embodiment, the software is 3D software. In anotherembodiment, the software is in the STL format. One of skill in the artwould be able to select suitable software for use herein including3DCrafter, 3DS Max, 3Dtin, Alibre, AC3D, Anim8or, Art of Illusion,AutoQ3D, AutoCAD, Blender, BRL-CAD, Cheetah3D, Cloud9, CreoElements/Direct, DrawPlus, FormZ, FreeCAD, GLC Player, Google SketchUp,K-3D, LeoCAD, Maya, Magics, MeshLab, NetFabb, OpenSCAD, Rhino3D,Solidworks, STL-viewer, Tinkercad, Wings 3D, ZBrush, among others. Theconstruct may be prepared from the top, bottom, or side as determined byone skilled in the art. In one embodiment, the construct is designedfrom the bottom.

The software may also be adapted to include code to modulate one or morecomponent of the bioprinter. In one embodiment, the software modulatesthe flow of gas into the cartridge. In another embodiment, the softwaremodulates the solenoid value that controls the flow of gas. In a furtherembodiment, the software controls the opening and closing of thesolenoid value that controls the gas flow.

Alternatively or in conjunction, the tissue construct may be designedvia reconstruction of tissues using medical imaging modalities. Examplesof medical imaging modalities include, without limitation, MagneticResonance Imaging (MRI) and Computed Tomography (CT).

J. NON-TRANSITORY COMPUTER READABLE STORAGE MEDIUM

The devices, systems, and methods may further include non-transitorycomputer readable storage media or storage media encoded with computerreadable program code. The computer readable storage medium may beconnected to a bioprinter or removable from a digital processing device.Examples of computer readable storage medium include CD-ROMs, DVDs,flash memory devices, solid state memory, magnetic disk drives, magnetictape drives, optical disk drives, cloud computing systems and services,among others.

K. COMPUTER MODULES

The devices, systems, and methods may include software, server, anddatabase modules. As known in the art, “computer module” is a softwarecomponent that interacts with a larger computer system, is one or morefiles and handles a specific task.

A computer module is optionally a stand-alone section of code or,optionally, code that is not separately identifiable. In someembodiments, the modules are in a single application. In otherembodiments, the modules are in a plurality of applications. In someembodiments, the modules are hosted on one machine. In otherembodiments, the modules are hosted on a plurality of machines. In someembodiments, the modules are hosted on a plurality of machines in onelocation. In other embodiments, the modules are hosted a plurality ofmachines in more than one location. Further described herein is theformatting of location and positioning data. In some embodiments, thedata files described herein are formatted in any suitable dataserialization format. A key feature of a computer module is that itallows an end user to use a computer to perform the identifiedfunctions.

L. GRAPHIC USER INTERFACE

The computer module may include a graphic user interface (GUI) whichprovides a picture and/or text and may be 2- or 3-dimensional. The GUImay be a touchscreen or multitouchscreen. The GUI may include a gridcomprising regularly spaced objects of substantially the same shape andsubstantially equal size.

The GUI may also be used to control one or more bioprinter parameter. Inone embodiment, the GUI is used to control one or more components of thebioprinter. In another embodiment, the GUI is used to control the EMR,deposition speed, and/or temperature of one or more component,environmental conditions of one or more component, optical device, amongothers.

M. COMPONENTS OF THE COMPOSITION

The tissues, organs, and vascular vessels may be prepared using thedevices, systems, and methods described herein together with acomposition. In one embodiment, the composition contains a biomaterialand optional additional components such as support material,non-cellular materials which enable bioprinting, or any combinationthereof.

The composition may be prepared by mixing the cells and a biocompatibleliquid or gel in a pre-determined ratio. The composition may optionallybe treating to facilitate extrusion onto the receiving means, increasedeposition efficiency, or initiate curing. In one embodiment, thecomposition is treated prior to extrusion to provide a desired celldensity, provide a desired viscosity, among others using techniquesknown in the art. Such methods which may be utilized to prepare thecomposition for extrusion include, without limitation, centrifugation,tangential flow filtration, electrical conductance, light, or anycombination thereof. The possible combinations of the components mayvary. However, the components do not need to be mixed into onecartridge.

(i) Biomaterial

In one embodiment, the biomaterial is a cell. The term “biomaterial”includes a composition (liquid, semi-solid, or solid) which containscells, proteins, genes, peptides, or any combination thereof. In oneembodiment, the biomaterial is viably maintained in a composition. Inanother embodiment, the biomaterial withstands the shear forces utilizedin the methods described herein. Any cell is suitable for use as thebiomaterial as determined by those skilled in the art. The compositionmay contain only one biomaterial or more than one biomaterial. In oneembodiment, the cell is a mammalian cell, plant cell, bacterial cell, orviral capsid.

Examples of cells include, without limitation, cell solutions, cellaggregates, cell suspensions, cell-comprising gels, multicellularbodies, tissues, or any combination thereof. A number of cells may beselected and include differentiated and undifferentiated cells. In oneembodiment the cells include, without limitation, contractile or musclecells (e.g., skeletal muscle cells, cardiomyocytes, smooth muscle cells,and myoblasts), connective tissue cells (e.g., bone cells, cartilagecells, fibroblasts, and cells differentiating into bone forming cells,chondrocytes, or lymph tissues), bone marrow cells, endothelial cells,skin cells, epithelial cells, breast cells, vascular cells, blood cells,lymph cells, neural cells, Schwann cells, gastrointestinal cells, livercells, pancreatic cells, lung cells, tracheal cells, corneal cells,genitourinary cells, kidney cells, reproductive cells, adipose cells,parenchymal cells, pericytes, mesothelial cells, stromal cells,undifferentiated cells (e.g., embryonic cells, stem cells, andprogenitor cells), endoderm-derived cells, mesoderm-derived cells,ectoderm-derived cells, and any combination thereof.

A “stem cell” as used herein refers to mitotic cells which candifferentiate into other cells. Stem cells may include, withoutlimitation, totipotent cells, pluripotent cells, multipotent cells,oligopotent cells, and unipotent cells. Stem cells may include embryonicstem cells, peri-natal stem cells, adult stem cells, amniotic stemcells, and induced pluripotent stem cells.

Accordingly, the methods and systems described herein are useful ingenerating tissue, organs, and vascular tubes. “Tissue” as used hereinrefers to a grouping of mammalian cells of the same type that perform aspecific function. Examples of tissues include, but are not limited to,connective (loose—areolar, reticular, and adipose and dense—regular andirregular), muscle (e.g., smooth, skeletal, and cardiac), nervous tissue(brain, spinal cord, and nerve), and epithelial (shape and arrangementclassified), and special connective (cartilage, bone, blood). In oneembodiment, intralumenal fluid perfusion may be used during thepreparation of vascular tubes to mimic blood pressures.

An “organ” is a collection of mammalian tissues in a specific structureto perform a function. Examples of organs include, but are not limitedto, skin, sweat glands, sebaceous glands, mammary glands, muscle,cartilage, bone marrow, bone, brain, hypothalamus, pituitary gland,pineal body, heart, blood vessels, cornea, heart valve, larynx, trachea,bronchus, lung, lymphatic vessel, salivary glands, mucous glands,esophagus, stomach, gallbladder, liver, pancreas, small intestine, largeintestine, colon, urethra, kidney, adrenal gland, conduit, ureter,bladder, fallopian tube, uterus, ovaries, testes, prostate, thyroid,parathyroid, meibomian gland, parotid gland, tonsil, adenoid, thymus,and spleen, teeth, gums, hair follicle, trachea, cartilage, or anycombination thereof.

A “vascular tube” as used herein refers to vessels or ducts that conveyfluids such as blood, lymph, water, or any combination thereof toanother location. The vascular tubes prepared as described herein haveuse in a variety of technologies including, without limitation, bypassgrafting, arteriovenous access, drug testing, cardiovascular devicetesting, and as stents. In one embodiment, the vascular tube is selectedfrom among arteries, elastic arteries, distributing arteries,arterioles, capillaries, venules, veins, large collecting vessels (suchas the subclavian vein, the jugular vein, the renal vein and the iliacvein) and venae cavae. In a further embodiment, the vascular tube has abranched structure. In another embodiment, the vascular tubes preparedas described herein may be of a thickness to withstand pressures whichare comparable to native physiological blood pressures. In a furtherembodiment, the vascular tubes may have an internal diameter of about0.5 to about 6 mm.

The cell density necessary for the composition may and is dependent onthe cells utilized and article fabricated using same. The cells may bepre-treated prior to incorporation into the composition using techniquessuch as incubation. The cell may also be at a selected temperature. Inone embodiment, the cells are frozen, maintained at a lower temperature,at ambient temperature, or at above ambient temperature. In oneembodiment, the cells are at about 37° C. or greater, depending on thetype of cell. In a further embodiment, embodiment, bacterial cells areat about 37° C. or greater. In another embodiment, the cells aremaintained at lower temperatures prior to, during or after printing.

(ii) Extrusion Agent

Ono or more extrusion agent may further be added to the compositiondescribed herein. In one embodiment, the extrusion agent cures, therebyencapsulating the biomaterial during formation of the fabricatedarticle. The term “cure” or variations as used herein is utilized todescribe the process for toughening or hardening one component of thecomposition described herein via the crosslinking of the components. Inone embodiment, the curing occurs concurrently as the bioprintingproceeds (i.e., the curing and bioprinting occur simultaneously). Thelength of time required for the curing to complete depends on thecomponents of the composition, article to be fabricated, and/orlaboratory conditions, among others. In one embodiment, curing iscomplete in less than about 1 year. In another embodiment, curing iscomplete in about 1 second to about 1 year. In a further embodiment,curing is complete in about 1 second to about 1 minute.

The extrusion agent may cure in the absence of exogenous agents ortechniques. In one embodiment, the extrusion agent is cured usingelectron beams, heat or chemical additives such as one or morephoto-initiator as described below. In a further embodiment, theextrusion agent is curable at a wavelength of about 405 nm or greater.

In one embodiment, the extrusion agent is a support material. Two ormore support materials, i.e., 2 to about 20, may be included in thecomposition. The support material is selected based on the desiredquality, viscosity, permeability, elasticity or hardness, adherency,biocompatibility, 3D printed structure, or the like. The supportmaterial is capable of hardening, viscous, excludes cells from growingor migrating into or adhering to it, or any combinations thereof. In oneembodiment, the support material is curable or cross-linkable at awavelength of about 405 nm or greater. The support material isoptionally removed prior to use of the fabricated article. In oneembodiment, the support material is removed via dissolution.Accordingly, the support material may be water-soluble, organic solventsoluble, dissolvable via enzymatic degradation, or dissolvable underacidic or basic conditions. In one embodiment, the enzymatic degradationis performed using a protease or lipase. The protease is, withoutlimitation, proteinase K, protease XIV, α-chymotrypsin, collagenase,matrix metalloproteinase-1 (MMP-1), MMP-2, or any combination thereof.The dissolution may alternatively be performed using cations or ions.

A variety of support materials may be selected by one skilled in the artusing the instant specification. In one embodiment, the support materialis a polymer. In another embodiment, the support material is athermoplastic polymer. In a further embodiment, the support material ispolyethylene oxide, poly-caprolactone, poly(L)-lactic acid (PLLA), orgelatin methacrylate, or any combination thereof. In yet anotherembodiment, the polymer is, without limitation, diacrylates such aspolyacrylic acid or polyethylene glycol diacrylate, methacrylates suchas hydroxyethyl methacrylate, norborenes, hydrogel, NovoGel™, gelatin,Matrigel™, hyaluronan, poloxamer, peptide hydrogel,poly(isopropyl-n-polyacrylamide), polydimethylsiloxane, polyacrylamide,polylactic acid, silicon, silk, surfactant polyols, thermo-responsivepolymers, hyaluronates, alginates, collagens, nanofibers,self-assembling nano fibers, hydrogels derived from collagen,hyaluronate, fibrin, agarose, chitosan, poly(ethylene oxide), polyvinylalcohol, polyphosphazene, or derivatives, copolymers or any combinationthereof. In yet a further embodiment, the diacrylate is PEG-DA. In stillanother embodiment, the methacrylate is PEG-MA. In a further embodiment,the norbornene is PEG-norbornene. In another embodiment, thepolyoxyethylene is poly(ethylene glycol). One of skill in the art wouldbe able to determine a suitable ratio of support material to cellsdepending on the other components of the composition.

(iii) Photo-Initiator

To create healthy 3D tissues, the damage to the cells by light(phototoxicity) must be minimized. Visible light reduces the energy thatthe tissues are exposed to. Thus, a photo-initiator also may be utilizedin the composition described herein. In one embodiment, thephoto-initiator promotes curing of the composition. In a furtherembodiment, the photo-initiator promotes cross-linking of one or morecomponent of the composition. In another embodiment, the photo-initiatoris a visible light photo-initiator. In a further embodiment, thephoto-initiator is activated when exposed to blue light. In anotherembodiment, the photo-initiator is lithiumphenyl-2,4,6-trimethylbenzoylphosphinate. In yet a further embodiment,the photo-initiator is the Irgacure™ 2959 product which contains one ormore of the following:

The ratio of the polymer to the photo-initiator is dependent on theselection of the polymer for use as described herein. The amount ofphoto-initiator must be sufficient to initiate cross-linking of thepolymer. In one embodiment, the weight ratio of the polymer to thephoto-initiator is about 1:1 to about 20:1.

(iv) Optional Components

The composition may optionally contain additional agents to facilitatepreparation of the desired product. One of skill in the art wouldreadily be able to select suitable additional agents for use herein.

In one embodiment, the composition includes an extracellular matrix.Examples of extracellular matrix components include, without limitation,collagen, fibronectin, laminin, hyaluronates, elastin, proteoglycans,gelatin, fibrinogen, fibrin, or any combination thereof. Thenon-cellular components of the composition may be retained or may beremoved prior to use using physical, chemical, or enzymatic means.

In a further embodiment, the composition includes a wetting agent asdescribed above.

In yet a further aspect, the composition includes a cell-binding factor.Examples of cell-binding factors useful herein include, withoutlimitation, fibronectin, lectins, cadherins, claudins, laminin, or anycombination thereof.

In another embodiment, the composition includes an antioxidant. Examplesof antioxidants include, without limitation, buffers such as phosphatebuffered saline.

In a further embodiment, the composition includes an agent that inhibitscell death. Examples of agents that inhibit cell death include thosethat inhibit the activity of an interleukin, interferon, granulocytecolony-stimulating factor, macrophage inflammatory protein, transforminggrowth factor B, matrix metalloproteinase, capsase, MAPK/JNK signalingcascade, Src kinase, Janus kinase, or any combination thereof.

In yet another embodiment, the composition includes an agent thatencourages cell adhesion. Examples of an agent that encourages celladhesion include, without limitation, Arginine-Glycine-Aspartic Acid(RGD), integrin, and extracellular matrix (ECM).

In still a further embodiment, the composition includespolyoxypropylenes and polyoxyethylenes.

In another embodiment, magnetic fields may be used to guide cellularreorganization and migration of the various cell types. Accordingly, thecompositions may contain magnetic particles such as ferromagneticnanoparticles, and are subjected to magnetic fields to guide cellularreorganization and migration.

A viscosity agent may optionally be added to the composition. By doingso, maintenance or fidelity of the extruded layer may be achieved due tothe imparted sufficient cohesive forces within the composition. In oneembodiment the selected viscosity agent depends on the shear thickeningor thinning of the components of the composition. In a furtherembodiment, the viscosity agent ensures that the composition issufficiently viscous to maintain its shape when extruded. In anotherembodiment, the viscosity agent ensures that the composition is not toothick so as to prevent its extrusion. In one embodiment, the viscosityagent is poly(ethylene oxide), gelatin, Pluronic F-127 (i.e., a(polyethyleneoxide)-(polypropyleneoxide)-(polyethyleneoxide) basedmaterial), hyaluronic acid, or any combination thereof.

N. FABRICATED ARTICLE

As discussed above, the methods, devices, and systems described hereinpermit the fabrication of a variety of articles using EMR at awavelength of about 405 nm or greater. Accordingly, the fabricatedarticle contains one EMR responsive material and cells as describedabove.

In one embodiment, the article is a cellular construct. In anotherembodiment, the article is 3-dimensional. In another embodiment, thearticle is a tissue construct such as an organ. In a further embodiment,the article is an array of cells. In still a further embodiment, thearticle is any body part (i.e., an organ) or organic structure toenhance and/or mediate bodily functions. In yet another embodiment, thearticle is a splint for implantation into a mammal, button (e.g., plug,stopgap, filling), among others.

The organ may be any component of a mammal. In one embodiment, the organis skin, sweat glands, sebaceous glands, mammary glands, bone, brain,hypothalamus, pituitary gland, pineal body, heart, blood vessels,larynx, trachea, bronchus, lung, lymphatic vessel, salivary glands,mucous glands, esophagus, stomach, gallbladder, liver, pancreas, smallintestine, large intestine, colon, urethra, kidney, adrenal gland,conduit, ureter, bladder, fallopian tube, uterus, ovaries, testes,prostate, thyroid, parathyroid, meibomian gland, parotid gland, tonsil,adenoid, thymus, spleen, teeth, gums, hair follicle, or cartilage.

A variety of plants or parts thereof may be printed using the methodsand systems described herein. In one embodiment, the plant is algae, aplant which produces a natural product, an agricultural plant designedfor human or animal ingestion, among others.

Bacteria and viral capsids may also be printed using the methods andsystems described herein. In one embodiment, the bacterium isEscherichia coli, streptococcus, Anaplasma, Basillus-brevis,Interrococcus, among others. In another embodiment, the viral capsid isAdeno-associated, Aichi, Australian bat lyssa, BK polyoma, Banna, Barmahforest, Bunyamwera, Bunya La Crosse, Bunya snowshoe hare, caudiovirales,Cercopithecine herpes, Chandipura, Chikungunya, Cosa A, Cowpox,Coxsackie, Crimean-Congo hemorrhagic fever, Dengue, Dhori, Dugbe,Duvenhage, Eastern equine encephalitis, Ebola, Echo,Encephalomyocarditis, Epstein-Barr, European bat lyssa, GB C/HepatitisG, Hantaan, Hendra, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis E,Hepatitis delta, Horsepox, Human adeno, Human astro, Human corona, Humancytomegalo, Human entero 68, 70, Human herpes 1, Human herpes 2, Humanherpes 6, Human herpes 7, Human herpes 8, Human immunodeficiency, Humanpapilloma 1, Human papilloma 2, Human papilloma 16,18, Humanparainfluenza, Human parvo B19, Human respiratory syncytial, Humanrhino, Human SARS corona, Human spumaretro, Human T-lymphotropic, Humantoro, Influenza A, Influenza B, Influenza C, Isfahan, JC polyoma,Japanese encephalitis, Junin arena, KI Polyoma, Kunjin, Lagos bat, LakeVictoria Marburg, Langat, Lassa, Lordsdale, Louping ill, Lymphocyticchoriomeningitis, Machupo, Mayaro, MERS corona, Measles, Mengoencephalomyocarditis, Merkel cell polyoma, Mokola, Molluscumcontagiosum, Monkeypox, Mumps, Murray valley encephalitis, New York,Nipah, Norwalk, O'nyong-nyong, Orf, Oropouche, Pichinde, Poli, Puntatoro phlebo, Puumala, Rabies, Rift valley fever, Rosa A, Ross river,Rota A, Rota B, Rota C, Rubella, Sagiyama, Sali A, Sandfly feversicilian, Sapporo, Semliki forest, Seoul, Simian foamy, Simian 5,Sindbis, Southampton, St. louis encephalitis, Tick-borne powassan,Torque teno, Toscana, Uukuniemi, Vaccinia, Varicella-zoster, Variola,Venezuelan equine encephalitis, Vesicular stomatitis, Western equineencephalitis, WU polyoma, West Nile, Yaba monkey tumor, Yaba-likedisease, Yellow fever, or Zika, among others.

The fabricated article may have a single layer or multiple layers,depending on the desired use, and may be unicellular or multicellular.The fabricated article may also include repeating pattern of functionalunits. The functional unit may have any suitable geometry, including,circles, squares, rectangles, triangles, polygons, and irregulargeometries. The repeating pattern of bioprinted functional units may bein the form of layers, i.e., a base layer and one or more layersthereon. The orientation of the layers is dependent on the final articleto be fabricated. In one embodiment, the layers may all be in the samedirection, may vary in direction, or any combination thereof. In anotherembodiment, the layers form a pattern. In a further embodiment, thelayers are alternating. In still another embodiment, the layers lack anypattern. FIG. 14 provides an illustration of a tissue fabricated usingthe methods described herein. FIG. 14A depicts a three-dimensional cellpatterning of a tissue-engineered construct. The three dimensionalstructure was composed of high strength synthetic polymer and a cellloaded hydrogel, fabricated by concurrent patterning of the twodifferent materials. As shown in FIGS. 14B and 14C, two differentmaterials, i.e., structural polymer 150 and cell loaded hydrogel 152,are concurrently deposited as a pattern to produce a single compositestructure. FIG. 14B depicts a cross sectional view demonstrating thelayer deposition. FIG. 14C depicts a top view demonstrating the latticestructure of the layered materials.

The fabricated article may be of any form which is useful to theattending clinician. In one embodiment, the fabricated article is a gelor solid.

The fabricated article may be formed by depositing a composition onto areceiving means. In one embodiment, the composition exits the orifice inthe form of a droplet or stream. As described above, the compositioncures after exposure to EMR of a wavelength of about 405 nm or greater.

Fabrication of the article may be continuous and/or substantiallycontinuous. In one embodiment, fabrication of the article is continuous.In another embodiment, fabrication of the article is continuous withperiods of inactivity. The fabricated article may be permitted sit for asufficient time after formation, i.e., incubated. In one embodiment, thefabricated article sits so as to permit cell adhesion, reorganizationmigration, or any combination thereof. Additional methods forfacilitating incubation may be performed utilized and include, withoutlimitation, heating, cooling, pressure, tension, compression, mechanicalforces, humidity changes, or any combination thereof.

At the end of the incubation period, the cells may be isolated byremoving any non-essential components. In one embodiment, any unwantedcomponents such as the support medium and/or extrusion agent is removed.In another embodiment, the support medium is physically removed awayfrom the support medium. In another embodiment, the support medium isremoved using water or any solvent that the non-cellular material issoluble in.

Finally, the fabricated article, lacking any non-essential components,may be finalized by permitting the cells to mature. In one embodiment,the fabricated article is place in a maturation chamber for growth.

O. KITS

Also provided are kits or packages containing any component of thebioprinter described herein. In one embodiment, the kit or packagecontains one or more of a printer stage, receiving means, cartridge,dispensing means such as a syringe, capillary tube, or pipette, aircompressor, EMR source, software, non-transitory computer readablestorage medium, computer module, graphic user interface, optical device,among others, or any combination thereof.

The kit or package may also include one or more component of thecomposition. In one embodiment, the kit or package contains one or morebiomaterial such as cells. In another embodiment, the kit or packagecontains one or more of an extrusion agent, photo-initiator,extracellular matrix, antioxidant, agent that inhibits cell death, agentthat encourages cell adhesion, magnetic particles, viscosity agent,extrusion agent such as support material, among others, or anycombination thereof.

The kit or package may further include one or more of a vial, tube,applicator, needle, dispensing means, lid, sealant, foil, and otherappropriate packaging and instructions for use.

The kit may contain one or more component of the composition. One ormore component of the composition may be separate, two or morecomponents may be combined, or any combination thereof. In oneembodiment, all of the components of the composition may be combined ina single dispensing means in the kit. In another embodiment, eachcomponent of the composition may be contained in a separate dispensingmeans in the kit. In a further embodiment, some of the components areindividually present in a dispensing means and some of the componentsare combined in a single dispensing means.

P. METHODS OF USING THE BIOPRINTERS

The bioprinters described herein and the fabricated synthetic, i.e.,man-made articles produced thereby have a variety of uses. In oneembodiment, the fabricated articles in the form of organs may betransplanted into a mammal in need thereof. The organs may betransplanted in the absence or presence of immunosuppressant agents asdetermined by the attending physician and transplanted organ. In oneembodiment, immunosuppressant agent may be administered, prior to,concurrently with, or subsequent to the transplantation. In anotherembodiment, the anti-rejection agent is an induction, maintenance,immunosuppressant. In a further embodiment, the anti-rejection agent is,without limitation, atgam, azathioprine, basiliximab, cyclosporine,daclizumab, methylprednisolone, mofetil, muromonab-CD3, mycophenolicacid, mycophenolate mofetil, OKT3, prednisone, rapamycin, sirolimus,tacrolimus, thymoglobulin, or any combination thereof. Additional agentsmay be administered prior to, concurrently with, and subsequent to thetransplantation and include, without limitation, pain medications, amongothers.

The fabricated synthetic articles produced as described herein also haveuse in testing a wide variety of chemical agents. By doing so, thenecessity to perform animal testing may be reduced or eliminated.Specifically, functions inherent to the particular cells of thefabricated articles may be evaluated, i.e., ensuring that the cells areproperly functioning. Such functions include, without limitation,protein function, cell marker viability, cell adhesion, or cellcontraction. Accordingly, the sensitivity, viability, toxicity, andresistance, among others, of the chemical agents may be evaluated.Accordingly, the fabricated synthetic articles produced herein have usein in vitro tests across a number of industries. The term “chemicalagent” as used herein refers to any single chemical or compositioncontaining that chemical agent which must be tested prior todistribution to the public. In one embodiment, the chemical agent may behousehold chemicals, pharmaceuticals such as antibiotics andchemotherapeutic agents, environmental agents, agricultural chemicals,food additives, healthcare agents, among others. In doing so, thechemical agent may be applied to a cellular structure prepared using thebioprinters herein. After application, the cellular structure may bemonitored. In one embodiment, the viability of the cells in the cellularstructure may be monitored and measured as necessary.

Q. EMBODIMENTS OF THE INVENTION

FIG. 15 illustrates one embodiment of a composition which utilizesviscosity agent 160, polymer 162, blue light photo-initiator 164, andcells 166 to provide tissue 168.

FIG. 16 depicts various possibilities of compositions for use inmultiple cartridges. If using a single cartridge, viscosity agent 160,polymer 162, photo-initiator 164, and cells 166 are mixed into singlesyringe 170. If using two cartridges, viscosity agent 160 is depositedseparately from the other ingredients, i.e., polymer 162,photo-initiator 164, and cells 166 using syringe 172. The contents ofsyringe 170 can be used in parallel across several cartridges.

R. EXAMPLES Example 1: Cell-Based Bowl

A cell-based construct was designed on the 3D CAD Solidworks™ softwareas a bowl material and exported as a standard tellellation language(stl) file. See, FIG. 17.

A polyethyelene glycol (PEG) based material (1000 MW) is combined withdeionized water at a 20% weight per volume ratio to form a solution.Polyethylene oxide at a 5.5% ratio with the PEG and lithiumphenyl-2,4,6-trimethylbenzoylphosphinate mixed at a 0.5% ratio with thePEG were then added to the polyethylene glycol solution. Humanmesenchymal stem cells were then pipetted into the combined solution andthe solution mixed for 1-2 minutes. The final mixture was then added toa syringe and placed within a cartridge of a bioprinter described above.The composition was extruded at a pressure of 275 kPa (40 psi) toprepare a bowl.

After printing, the bowl was incubated for 8 hours under cell culturemedia at 37° C. A cell viability assay was conducted using a live/deadkit assay from LifeTechnologies and illustrated that about 90% of thecells were alive. See, FIG. 18.

Example 2: Synthetic Ear

A cell-based construct was designed on the 3D CAD Solidworks™ softwareas an ear and exported as an stl file. See, FIG. 19.

A polyethyelene glycol based material (1000 MW) is combined withdeionized water at a 20% weight per volume ratio to form a solution.Polyethylene oxide at a 5.5% ratio with the PEG and lithiumphenyl-2,4,6-trimethylbenzoylphosphinate mixed at a 0.5% ratio with thePEG were then added to the polyethylene glycol solution. Humanmesenchymal stem cells were then pipetted into the combined solution andthe solution mixed for 1-2 minutes. The final mixture was then added toa syringe and placed within a cartridge of a bioprinter described above.The composition was extruded at a pressure of 275 kPa (40 psi) toprepare an ear.

After printing, the ear was incubated for 8 hours under cell culturemedia at 37° C. A cell viability assay was conducted using a live/deadkit assay from LifeTechnologies and illustrated that about 90% of thecells were alive. See, FIG. 20.

When ranges are used herein, all combinations, and subcombinations ofranges for specific embodiments therein are intended to be included.

The disclosures of each patent, patent application, and publicationcited or described in this document are hereby incorporated herein byreference, in its entirety.

Those skilled in the art will appreciate that numerous changes andmodifications can be made to the preferred embodiments of the inventionand that such changes and modifications can be made without departingfrom the spirit of the invention. It is, therefore, intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the invention.

1. A three-dimensional bioprinter comprising: a cartridge-extruderassembly comprising: a cartridge for receiving and holding a compositioncomprising biomaterial that cures after exposure to electromagneticradiation (EMR) at or above 405 nm; and an extruder configured to engagewith the cartridge and extrude the composition; a receiving platepositioned below the cartridge configured to receive the composition;and an EMR module that emits EMR at a wavelength of about 405 nm orhigher, wherein the EMR module is positioned adjacent to thecartridge-extruder assembly, independent of the cartridge, andconfigured to emit EMR towards at least one of the receiving plate andthe cartridge.
 2. The three-dimensional bioprinter of claim 1, whereinthe cartridge-extruder assembly is configured to extrude simultaneouslyas the EMR module emits the EMR.
 3. The three-dimensional bioprinter ofclaim 1, wherein the EMR module is configured to emit EMR for about 1 toabout 5 seconds.
 4. The three-dimensional bioprinter of claim 1, whereinthe extruder comprises a syringe, capillary tube, or micropipette. 5.The three-dimensional bioprinter of claim 1, wherein at least one of thecartridge-extruder assembly and the receiving unit comprises atemperature control unit capable of adjusting temperatures to betweenabout −10 to 300° C.
 6. The three-dimensional bioprinter of claim 5,wherein the temperature control unit comprises at least one of a Peltierheating and cooling unit.
 7. The three-dimensional bioprinter of claim1, wherein said bioprinter comprises a first opening which permitsinsertion of said cartridge and a second opening which permits a portionof said cartridge to exit said bioprinter.
 8. The three-dimensionalbioprinter of claim 7, wherein said cartridge has a capacity of about 1μL to about 100 mL and diameter of about 1 μm to about 10 cm.
 9. Thethree-dimensional bioprinter of claim 1, wherein said composition isdispensed using pressure generated using at least one of a piston,compressed gas, hydraulics, air compressor, piezo-electronics, andinkjet dispensing extrusions.
 10. The three-dimensional bioprinter ofclaim 1, comprising two or more cartridges.
 11. The three-dimensionalbioprinter of claim 10, wherein each of said cartridge is attached toeach other.
 12. The three-dimensional bioprinter of claim 10, whereineach of said cartridge is positioned separately from each other.
 13. Thethree-dimensional bioprinter of claim 10, wherein each of said cartridgecontains a different composition.
 14. The three-dimensional bioprinterof claim 10, wherein said cartridge contains the same composition. 15.The three-dimensional bioprinter of claim 10, wherein a first cartridgeextrudes a first material onto the receiving plate and an EMR modulepositioned adjacent to a second cartridge cures the extruded firstmaterial.
 16. The three-dimensional bioprinter of claim 1, wherein saidcomposition further comprises at least one of an extrusion agent whichis curable at a wavelength of about 405 nm or greater, aphoto-initiator, viscosity agent, and a biocompatible agent.
 17. Thethree-dimensional bioprinter of claim 16, wherein said extrusion agentis at least one of polyoxyalkylene, diacrylate, methacrylate,norbornene, gelatin, methacrylate, methacrylated hyaluronic acid,hydroxyethyl-methacrylate-derivatized-dextran, p(HPMAm-lactate)-PEG,gold nanorods, carbon nanotubes, collagen, polyethylene oxide,poly-caprolactone, and poly(L)-lactic acid.
 18. The three-dimensionalbioprinter of claim 16, wherein said photo-initiator is lithiumphenyl-2,4,6-trimethylbenzoylphosphinate or one or more of thefollowing:


19. The three-dimensional bioprinter of claim 16, wherein said viscosityagent is at least one of poly(ethylene oxide), gelatin, Pluronic F-127,and hyaluronic acid.
 20. The three-dimensional bioprinter of claim 16,wherein the biocompatible agent comprises cells.